A method for catalytic synthesis of ethyl rutin by using a eutectic solvent

Abstract

The application discloses a method for synthesizing ethyl rutin by using a eutectic solvent, and belongs to the technical field of green chemical synthesis. The method comprises the following steps: uniformly mixing reaction raw materials, a eutectic solvent solution, a rutinase solution and anhydrous ethanol, and performing enzymolysis reaction to obtain an enzymolysis solution; then, purification is performed to obtain ethyl rutin; wherein the reaction raw materials are one or both of kaempferol-rutin and rutin; and the eutectic solvent in the eutectic solvent solution is one of betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, choline chloride-urea-acetamide. Compared with a conventional synthesis method of ethyl rutin, the enzymatic catalysis system composed of betaine-ethylene glycol can increase the relative efficiency of ethyl rutin synthesis to 264.87%.

Description

TECHNICAL FIELD

[0001] The application relates to a method for catalytically synthesizing ethyl rutin by using a eutectic solvent, and belongs to the technical field of green chemical synthesis. BACKGROUND

[0002] Ethyl rutin is an important hypoglycemic active ingredient and is expected to be used in the field of hypoglycemia.

[0003] At present, ethyl rutin is synthesized in an environment of low-concentration ethanol aqueous solution by means of enzyme catalysis. Specifically, the reaction system mainly comprises rutin, rutin-degrading enzyme (which can be separated from tartary buckwheat) and low-concentration ethanol aqueous solution, rutin serves as a donor of a rutin group, the rutin group is transferred to an ethanol acceptor through the transglycosylation of the rutin-degrading enzyme, and finally ethyl rutin is formed. However, the synthesis method has the following problems: first, rutin, as a flavonoid, has low solubility in the ethanol aqueous solution, when the concentration of rutin is greater than 2 mg / mL, a large amount of precipitate is generated, leading to low reaction efficiency in the subsequent synthesis reaction process; second, ethanol as an organic solvent has an adverse effect on the activity of the enzyme, affecting the enzyme catalytic activity and the reaction efficiency.

[0004] As a new type of green solvent, the eutectic solvent has high solubility for lipid-soluble components and can avoid the damage of conventional organic reagents to the active center of the enzyme. Based on the above characteristics, the eutectic solvent is widely used in the extraction process of active compounds (such as flavonoids, polyphenols, carotenoids, etc.) in food, Chinese herbal medicines and other raw materials, but there is no report on the application of the eutectic solvent to the synthesis of ethyl rutin. SUMMARY

[0005] [Technical problem]

[0006] The method for synthesizing ethyl rutin by using low-concentration ethanol aqueous solution and enzyme assistance has poor solubility and low enzyme activity, leading to low yield and purity.

[0007] The eutectic solvent has not been used for synthesizing ethyl rutin.

[0008] [Technical solution]

[0009] In order to solve the above problems, the eutectic solvent is used in the catalytic system of ethyl rutin, and the green and efficient preparation of ethyl rutin is realized.

[0010] The first object of the application is to provide a method for catalytically synthesizing ethyl rutin by using a eutectic solvent, comprising the following steps:

[0011] The reaction raw material, the eutectic solvent solution, the rutin-degrading enzyme solution and anhydrous ethanol are uniformly mixed to perform an enzymatic reaction, and an enzymatic solution is obtained; then, purification is performed to obtain ethyl rutin.

[0012] The reaction raw material is one or both of kaempferol-rutin and rutin.

[0013] The eutectic solvent in the eutectic solvent solution is one of betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, and choline chloride-urea-acetamide.

[0014] In an embodiment of the present application, the molar ratio of betaine to ethylene glycol in the betaine-ethylene glycol is 1:1.5-2.5.

[0015] In an embodiment of the present application, the molar ratio of choline chloride to urea in the choline chloride-urea is 1:1.5-2.5.

[0016] In an embodiment of the present application, the molar ratio of choline chloride to malic acid in the choline chloride-malic acid is 1:0.5-1.5.

[0017] In an embodiment of the present application, the molar ratio of choline chloride to urea to acetamide in the choline chloride-urea-acetamide is 1:0.5-1.5:0.5-1.5.

[0018] In an embodiment of the present application, the ratio of the reaction raw material, the eutectic solvent solution, the rutin-degrading enzyme solution, and the anhydrous ethanol is 1g:3-45mL:0.5-1.5mL:0.5-5mL.

[0019] In an embodiment of the present application, the solvent of the eutectic solvent solution is water, and the volume fraction is 15-25%.

[0020] In an embodiment of the present application, the volume fraction of the anhydrous ethanol in the total reaction system is 5-50%.

[0021] In an embodiment of the present application, the rutin-degrading enzyme aqueous solution is prepared by dissolving rutin-degrading enzyme powder in water, and the concentration is 0.5-1.5mg / mL.

[0022] In an embodiment of the present application, the enzyme activity of the rutin-degrading enzyme is 15-30U / mg, and the preparation method of the rutin-degrading enzyme is as follows: defatted tartary buckwheat powder is mixed with acetic acid buffer, extracted overnight, frozen and centrifuged, and the supernatant is the crude enzyme solution of the rutin-degrading enzyme; then the rutin-degrading enzyme is separated, extracted, concentrated, and dried.

[0023] In an embodiment of the present application, the enzymatic reaction is carried out at pH 4-8 and 20-50℃ for 1-60min.

[0024] In one embodiment of the present application, the purification is column chromatography, amino column chromatography; wherein the column chromatography is macroporous adsorption resin D101 column chromatography, eluted with water, discarding the first 1.5 times column volume of water eluate, then eluted with 2 times column volume of water, and the concentrated ethyl rutin crude product is collected; the amino column chromatography is amino column chromatography of the ethyl rutin crude product with 75% acetonitrile aqueous solution as the eluent, and the pure ethyl rutin is obtained after concentration of the eluate collected from the 5th to 8th column volume.

[0025] A second object of the present application is the ethyl rutin prepared by the method of the present application.

[0026] A third object of the present application is the use of the ethyl rutin of the present application in the field of food or medicine.

[0027] [Advantages]

[0028] (1) The eutectic solvent used in the present application is a green solvent with better solubility and can avoid the damage of conventional organic reagents to enzyme activity.

[0029] (2) Compared with the conventional synthesis method of ethyl rutin, the relative efficiency of the synthesis of ethyl rutin by the betaine-ethylene glycol enzyme catalytic system used in the present application can be increased to 264.87%.

[0030] (3) After conversion by the betaine-ethylene glycol enzyme catalytic system used in the present application, the yield of ethyl rutin is 3.24-3.76 times that of the conventional system. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The synthesis route of ethyl rutin in the present application is shown in the figure.

[0032] Figure 2 The HPLC-MS total ion current chromatogram of Example 2 is shown in the figure.

[0033] Figure 3 The HPLC-MS total ion current chromatogram (A) and mass spectrum information chart (B) of the product in Example 2 are shown in the figure.

[0034] Figure 4 The HPLC-MS total ion current chromatogram of Example 3 is shown in the figure.

[0035] Figure 5 The HPLC-MS total ion current chromatogram of Comparative Example 1 is shown in the figure.

[0036] Figure 6 The HPLC-MS total ion current chromatogram of Comparative Example 2 is shown in the figure. DETAILED DESCRIPTION

[0037] The following describes preferred embodiments of the present application, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0038] Test method:

[0039] Quantitative method of ethylrutin:

[0040] Instrument: ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometer (UPLC-Q-TOF-MS, Waters, USA);

[0041] Chromatographic column: CORTECS C18+(2.7 μm, 2.1 x 150 mm);

[0042] Liquid phase condition:

[0043] Phase A: acetonitrile; Phase B: 0.1% formic acid water; flow rate: 0.3 mL / min; column temperature: 45℃;

[0044] Gradient elution program: initial condition of B phase 100%, B phase changed from 100% to 90% within 1 min to 5 min, B phase changed from 90% to 50% within 5 min to 6 min, B phase changed from 50% to 100% within 6 min to 7 min, B phase remained 100% after 7 min, running time 10 min;

[0045] The content of ethylrutin was calculated by the standard curve.

[0046] Raw materials used in the examples:

[0047] Rutin and kaempferol-rutin standard were purchased from Yelv Biotechnology Co., Ltd.;

[0048] Rutin degrading enzyme: prepared according to the literature (Zhang YW, Li J, Yuan Y, et al. Purification, enzymatic properties and partial primary structure analysis of rutin degrading enzyme from tartary buckwheat seeds. Bioengineering Bulletin, 2017, 33(5): 796-807), and the enzyme activity was 20.84 U / mg;

[0049] Preparation of deep eutectic solvent:

[0050] The hydrogen bond donor and the hydrogen bond acceptor were mixed in a reaction bottle at a proper molar ratio (Table 1) and placed in a magnetic stirrer at 80℃ until a uniform liquid was formed.

[0051] Table 1

[0052] No. Hydrogen donor Hydrogen acceptor 1 Hydrogen acceptor 2 Molar ratio 1 Choline chloride Urea 1:2 2 Choline chloride Glycerol 1:2 3 Choline chloride Ethylene glycol 1:2 4 Choline chloride Propylene glycol 1:2 5 Choline chloride Citric acid 1:1 6 Choline chloride Malic acid 1:1 7 Choline chloride Lactic acid 1:1 8 Choline chloride Formic acid 1:2 9 Choline chloride Acetic acid 1:2 10 Choline chloride Urea Glycerol 1:1:1 11 Choline chloride Urea Ethylene glycol 1:1:1 12 Choline chloride Urea Acetamide 1:1:1 13 Choline chloride Urea Thiourea 1:1:1 14 Choline chloride Ethylene glycol Acetamide 1:1:1 15 Choline chloride Ethylene glycol Thiourea 1:1:1 16 Choline chloride Propylene glycol Glycerol 1:1:1 17 Choline chloride Propylene glycol Acetamide 1:1:1 18 Choline chloride Propylene glycol Thiourea 1:1:1 19 Choline chloride Propylene glycol Urea 1:1:1 20 Choline chloride Formic acid Acetic acid 1:1:1 21 Choline chloride Acetic acid Glycerol 1:1:1 22 Choline chloride Acetic acid Urea 1:1:1 23 Betaine Lactic acid 1:1 24 Betaine Ethylene glycol 1:2 25 Menthol Lauryl acid 1:2

[0053] Example 1 Screening process of deep eutectic solvent

[0054] A method for catalytic synthesis of ethyl rutin by using a eutectic solvent, comprising the following steps:

[0055] 1g of rutin, 44mL of a 20% eutectic solvent (Table 1) aqueous solution, 1mL of a 1mg / mL rutin-degrading enzyme aqueous solution, and 5mL of anhydrous ethanol were mixed uniformly (to make the final concentration of anhydrous ethanol in the system 10%), and an enzymatic reaction was carried out at pH 5 and 40℃ for 1min (short time for reaction rate); immediately, the reaction was placed in a boiling water bath to terminate the enzymatic reaction;

[0056] The ethyl rutin content refers to the concentration of ethyl rutin in the system after the reaction is completed;

[0057] The blank group was replaced with a 10% ethanol aqueous solution instead of the eutectic solvent, and the ethyl rutin content in the system after the reaction was 100%; the relative synthesis efficiency of the blank group was used to represent the relative synthesis efficiency of each eutectic solvent treatment group:

[0058] The relative synthesis efficiency of ethyl rutin (%) = the ethyl rutin content of the eutectic solvent treatment group / the ethyl rutin content of the blank group x 100%;

[0059] The specific results are shown in Table 2: from Table 2, it can be seen that the relative synthesis efficiency of the enzyme-catalyzed system composed of betaine-ethylene glycol can be increased to 264.87%; the relative synthesis efficiency of the enzyme-catalyzed system composed of choline chloride-urea can also be increased (more than 100%).

[0060] Table 2

[0061]

[0062]

[0063] Example 2

[0064] A method for catalytic synthesis of ethyl rutin by using a eutectic solvent, comprising the following steps:

[0065] 1g of rutin, 39mL of a 15% betaine-ethylene glycol aqueous solution, 1mL of a 1mg / mL rutin-degrading enzyme aqueous solution, and 10mL of anhydrous ethanol were mixed uniformly (to make the final concentration of anhydrous ethanol in the system 20%), and an enzymatic reaction was carried out at pH 4 and 25℃ for 30min to obtain an enzymatic hydrolysate;

[0066] The enzymatic hydrolysate was subjected to column chromatography on a macroporous adsorption resin D101 column, eluted with water, and the first 1.5 times the column volume of water eluate was discarded, and then 2 times the column volume of water was used for elution, and the ethyl rutin crude product was collected and concentrated;

[0067] Crude ethylrutin was subjected to amino column chromatography using a 75% (v / v) acetonitrile aqueous solution as the eluent. The eluent from the 5th to the 8th column volumes was collected and concentrated to obtain pure ethylrutin.

[0068] Example 3

[0069] A method for synthesizing ethylrutin using a eutectic solvent as a catalyst includes the following steps:

[0070] Mix 100g of kaempferol-rutin, 350mL of 20% betaine-ethylene glycol solution (solvent is water), 100mL of 1mg / mL rutin-degrading enzyme aqueous solution, and 300mL of anhydrous ethanol evenly (so that the final volume concentration of anhydrous ethanol in the system reaches 40%), and carry out the enzymatic hydrolysis reaction at pH 8 and 30℃ for 30min to obtain the enzymatic hydrolysate;

[0071] The enzymatic hydrolysate was subjected to macroporous adsorption resin D101 column chromatography, eluted with water, and the first 1.5 column volumes of water eluent were discarded. Then, the eluent was eluted with 2 column volumes of water, and the crude ethylrutin was collected and concentrated.

[0072] Crude ethylrutin was subjected to amino column chromatography using a 75% (v / v) acetonitrile aqueous solution as the eluent. The eluent from the 5th to the 8th column volumes was collected and concentrated to obtain pure ethylrutin.

[0073] Comparative Example 1

[0074] The betaine-ethylene glycol solution in Example 2 was changed to water, while other aspects remained the same as in Example 2, to obtain ethyl rutin.

[0075] Comparative Example 2

[0076] The betaine-ethylene glycol solution in Example 3 was changed to water, while other aspects remained the same as in Example 3, to obtain ethyl rutin.

[0077] The enzymatic hydrolysates and ethyl rutin obtained in Examples 2 and 3 and Comparative Examples 1 and 2 were subjected to performance tests. The test results are shown in Table 3 below. Figures 2-6 :

[0078] from Figure 2 and Figure 4 The comparison shows that the peak area in the ion chromatogram of Example 2 is much larger than that in Comparative Example 1, which means that the content of ethyl rutin in the enzyme reaction extract of Example 2 is much larger than that in Comparative Example 1.

[0079] from Figure 3 It can be seen that the substance prepared in Example 2 is indeed ethylrutin;

[0080] from Figure 5 and Figure 6 No. Hydrogen donor Hydrogen acceptor 1 Hydrogen acceptor 2 Molar ratio Choline chloride Urea Choline chloride Glycerol Choline chloride Ethylene glycol Choline chloride Propylene glycol Choline chloride Citric acid Choline chloride Malic acid Choline chloride Lactic acid Choline chloride Formic acid Choline chloride Acetic acid Choline chloride Urea Glycerol Choline chloride Urea Ethylene glycol Choline chloride Urea Acetamide Choline chloride Urea Thiourea Choline chloride Ethylene glycol Acetamide Choline chloride Ethylene glycol Thiourea Choline chloride Propylene glycol Glycerol Choline chloride Propylene glycol Acetamide Choline chloride Propylene glycol Thiourea Choline chloride Propylene glycol Urea Choline chloride Formic acid Acetic acid Choline chloride Acetic acid Glycerol Choline chloride Acetic acid Urea Betaine Lactic acid Betaine Ethylene glycol Menthol Lauryl acid Figures 2-6 Figure 2 Figure 4 Figure 3 Figure 5 Figure 6 No. Hydrogen donor Hydrogen acceptor 1 Hydrogen acceptor 2 Molar ratio Choline chloride Urea Choline chloride Glycerol Choline chloride Ethylene glycol Choline chloride Propylene glycol Choline chloride Citric acid Choline chloride Malic acid Choline chloride Lactic acid Choline chloride Formic acid Choline chloride Acetic acid Choline chloride Urea Glycerol Choline chloride Urea Ethylene glycol Choline chloride Urea Acetamide Choline chloride Urea Thiourea Choline chloride Ethylene glycol Acetamide Choline chloride Ethylene glycolIt can be seen from the comparison that the peak area in the ion flow chart of Example 3 is much larger than that of Comparative Example 2, which means that the content of ethyl rutin in the enzyme reaction extract of Example 3 is much larger than that of Comparative Example 2;

[0081] The conclusion is consistent with Table 3.

[0082] Table 3

[0083]

[0084] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for synthesizing ethylrutin using a eutectic solvent as a catalyst, characterized in that, Includes the following steps: The reactants, eutectic solvent solution, rutin-degrading enzyme solution, and anhydrous ethanol were mixed evenly and subjected to enzymatic hydrolysis to obtain the hydrolysate; then purified to obtain ethyl rutin. The reaction raw materials are one or two of kaempferol-rutin and rutin. The eutectic solvent in the eutectic solvent solution is one of betaine-ethylene glycol, choline chloride-urea, choline chloride-malic acid, or choline chloride-urea-acetamide; In the betaine-ethylene glycol mixture, the molar ratio of betaine to ethylene glycol is 1:1.5-2.

5. The molar ratio of choline chloride to urea in choline chloride-urea solution is 1:1.5-2.

5. In the choline chloride-malic acid mixture, the molar ratio of choline chloride to malic acid is 1:0.5-1.

5. The molar ratio of choline chloride, urea, and acetamide in the choline chloride-urea-acetamide mixture is 1:0.5-1.5:0.5-1.

5. The ratio of reaction raw materials, eutectic solvent solution, rutin-degrading enzyme solution, and anhydrous ethanol is 1g: 3-45mL: 0.5-1.5mL: 0.5-5mL; The enzymatic hydrolysis reaction is carried out at pH 4-8 and 20-50℃ for 1-60 minutes.

2. The method according to claim 1, characterized in that, The volume fraction of anhydrous ethanol in the total reaction system is 5-50%.

3. The method according to claim 1, characterized in that, The solvent for the eutectic solvent solution is water, with a volume fraction of 15-25%.

4. The method according to claim 1, characterized in that, The rutin-degrading enzyme aqueous solution is prepared by dissolving rutin-degrading enzyme powder in water.

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